insulin regulation of human ovarian androgensandrogens and decreasing circulating sex...

Insulin regulation of human ovarian androgensJohn E.Nestler

Division of Endocrinology and Metabolism, Department of Internal Medicine, and Department ofObstetrics and Gynecology, Medical College of Virginia/Virginia Commonwealth University,

Richmond, VA 23298, USA

Hyperinsulinaemic insulin resistance is charac-teristic of many, if not all, women with poly cysticovary syndrome (PCOS). We will review evid-ence suggesting that hyperinsulinaemia pro-motes hyperandrogenism in PCOS by twodistinct and independent mechanisms: (i) byincreasing circulating ovarian androgens; and(ii) by directly reducing serum sex hormone-binding globulin concentrations. The net resultof these actions is to increase circulating freetestosterone concentrations. It appears likelythat an inherent (probably genetically deter-mined) ovarian defect need be present in womenwith PCOS, which makes the ovary susceptibleto insulin stimulation of androgen production.Limited evidence suggests that hyperinsulin-aemia might also promote ovarian androgenproduction by influencing pituitary release ofgonadotrophins. This latter possibility, however,has not been critically evaluated. The clinicalimplication of these findings is that ameliorationof hyperandrogenism in women with PCOS maybe achieved by interventions which improveinsulin sensitivity and reduce circulating insulin.Such measures might include, but are not lim-ited to, weight loss, dietary modification, andinsulin-sensitizing medications.Key words: androgen/insulin/metabolism/ovary/steroid

IntroductionPolycystic ovary syndrome (PCOS) has beendefined clinically by hyperandrogenism and ano-vulation. Recently, it has become apparent that

hyperinsulinaemic insulin resistance is also a prom-inent feature of both obese and non-obese womenwith PCOS, and increasing evidence suggests thathyperinsulinaemia plays a pivotal role in the patho-genesis of this disorder (Nestler and Strauss, 1991;Nestler, 1994).

We will review evidence indicating that insulincontributes to the hyperandrogenism of PCOS byboth increasing serum concentrations of ovarianandrogens and decreasing circulating sex hormone-binding globulin (SHBG) concentrations. Althoughevidence exists that insulin may also influencefolliculogenesis directly, this area will not bereviewed here and has been discussed elsewhere(Nestler, 1994).

Insulin resistance: an integral feature ofPCOS

The evidence is overwhelming that PCOS is adisorder characterized by insulin resistance and acompensatory hyperinsulinaemia. Both obese andnon-obese women with PCOS have been shownto be more insulin-resistant and hyperinsulinaemicthan age- and weight-matched normal women(Burghen et al, 1980; Chang et al, 1983; Pasqualiet al, 1983; Shoupe et al, 1983; Geffner et al,1986; Stuart etal, 1986; Dunaif etal, 1987, 1989,1990, 1992; Ciaraldi et al, 1992; Dahlgren et al,1992; Rosenbaum et al, 1993; Ehrmann et al,1995). It is noteworthy that insulin resistanceand hyperinsulinaemia appear to be characteristicfeatures of PCOS not only in the US, but in othersocieties as well. For example, women with PCOSfrom the US, Japan and Italy were comparedwith their respective normal counterparts (Carmina

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et al, 1992). Clinical differences existed amongthese women, and women with PCOS from theUS were significantly more obese than Japanesewomen with PCOS. Moreover, women with PCOSfrom the US and Italy were hirsute, whereas womenwith PCOS from Japan were not. Nonetheless,regardless of these differences, women with PCOSfrom all three countries manifested insulin resist-ance and hyperinsulinaemia. This common findingacross multiple ethnic groups suggests that hyper-insulinaemic insulin resistance represents a univer-sal feature of PCOS.

It is unlikely that the hyperinsulinaemic insulinresistance of PCOS occurs as a result of hyperand-rogenism. Insulin resistance persists in womenwith PCOS who have undergone either subtotal(Imperato-McGinley et al, 1978) or total(Nagamani et al, 1986) removal of the ovaries, orin whom ovarian androgen production has beensuppressed with the use of a long-acting gonadotro-phin-releasing hormone (GnRH) agonist (Geffneret al, 1986; Dunaif et al, 1990; Nestler et al,1991a). Prepubertal women with acanthosisnigricans are hyperinsulinaemic, yet elevatedserum androgen concentrations do not appear untilseveral years following the diagnosis of insulinresistance (Richards et al, 1985). Some womenwith point mutations in the insulin receptor genecausing hyperinsulinaemic insulin resistance havebeen shown to have PCOS (Moller and Flier, 1988;Yoshimasa et al, 1988; Kim et al, 1992). Finally,normal men have androgen concentrations 10-30-fold higher than women, yet they do notdemonstrate insulin resistance. Collectively, theseobservations support the notion that the hyperinsul-inaemia of PCOS is a causal factor in the accom-panying hyperandrogenism.

Insulin and ovarian androgens

Effects of insulin on circulating ovarianandrogens

Human ovaries possess insulin receptors (Poretskyet al, 1984, 1985; Jarrett et al, 1985) suggestinga role for this peptide in the regulation of ovarianfunction, and in-vitro studies have demonstratedthat insulin can directly stimulate androgen produc-tion by ovarian stroma obtained from women with

PCOS (Barbieri et al, 1986). Moreover, insulinand insulin-like growth factor I (IGF-I) augmentluteinizing hormone (LH)-stimulated androgen bio-synthesis in rat ovarian theca cells (Cara andRosenfield, 1988; Cara et al, 1990).

It has been substantially more difficult to demon-strate the effect of insulin on ovarian androgensin vivo. Multiple studies have been conductedwhere serum testosterone was monitored in womenduring an acute elevation of circulating insulin,and the results in women with PCOS appear tobe confusing and conflicting. Serum testosteroneeither rose, did not change, or fell in the womenwith PCOS (Nestler et al, 1987; Smith et al,1987; Stuart etal, 1987; Micic etal, 1988; Dunaifand Graf, 1989).

There are several problems associated with insu-lin infusion studies that may account for theconflicting results reported in the literature. Firstof all, the very nature of these studies dictates thatthe duration of insulin elevation is brief, lastingonly a few hours. Furthermore, in many studiesthe degree of insulin elevation was far abovethe physiological range. Finally, several of thesestudies did not control for the volume of fluidsinfused, diurnal and day-to-day variations in steroidconcentrations, or for unmeasured perturbationssuch as the well described increase in catecho-lamines that accompanies insulin infusions (Roweetal, 1981).

Perhaps the single situation in which investig-ators induced long-term hyperinsulinaemia whilemonitoring serum androgen concentrations was thecase study reported by DeClue et al. (1991). Theseinvestigators cared for a young woman with PCOSwho was diabetic and manifested a high degree ofinsulin resistance. High-dose insulin therapy wasstarted, and marked hyperinsulinaemia was main-tained over several months. During this period ofinsulin elevation, circulating testosterone concen-trations progressively rose and ovarian volume (asmeasured by ultrasound) increased 2-fold. Whenthe insulin infusion was discontinued and seruminsulin concentrations began to fall, serum testos-terone concentrations fell as well, eventually fallinginto the normal range. A clear concordance existedbetween serum insulin and testosterone concentra-tions, suggesting a cause-and-effect relationship.

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These findings strongly suggested that insulinstimulated ovarian androgen production in thiswoman, and was directly responsible for the hyper-androgenism.

In order to study the role of physiologicalelevations of insulin in PCOS while avoidingproblems associated with insulin infusions, weutilized the drug diazoxide (Proglycem, MedicalMarket Specialties, Boonton, NJ, USA) to suppressinsulin release from the pancreas. When five obesewomen with PCOS were administered diazoxidefor 10 days, the fasting serum insulin concentrationwas suppressed, serum glucose rose, and the insulinresponse to an oral glucose challenge decreasedmarkedly (Nestler et al, 1989). More importantly,serum total testosterone concentrations fell in allfive women with PCOS during this period ofinsulin suppression by diazoxide. Mean serum totaltestosterone fell by 17% from 2.5 ± 0.4 nmol/1 to2.1 ± 0.3 nmol/1 (P <0.007). Moreover, serumSHBG levels rose during diazoxide administrationfrom a mean value of 13.2 ± 1.0 nmol/1 to 21.7± 4 . 1 nmol/1, but this elevation did not attainstatistical significance (P = 0.09). Because of theconcurrent fall in serum total testosterone and risein SHBG concentrations, serum-free (i.e. non-SHBG-bound) testosterone concentrations fell by28% from 0.19 ± 0.03 nmol/1 to 0.14 ± 0.02nmol/1 (P <0.01; Figure 1).

To verify that this decline in circulating testos-terone was not due to diazoxide itself, as wellas determining whether insulin regulates ovarianandrogens in normal women, a control group offive non-obese healthy women was studied in anidentical manner (Nestler et al, 1990). In markedcontrast with the obese women with PCOS,diazoxide treatment altered neither serum testos-terone (P = 0.71) nor SHBG (P = 0.24) concentra-tions in the non-obese healthy women with normalamounts of circulating insulin.

Subsequent to these findings, the insulin-sensit-izing drug metformin (Glucolab, Lab. Relab,Caracas, Venezuela) was administered to womenwith PCOS in three independent studies (Velazquezet al, 1994; Crave et al, 1995; Nestler andJakubowicz, 1996). In the first report (Velazquezet al, 1994), treatment of 26 PCOS women withmetformin for 8 weeks resulted in decreases in

both total and free testosterone and a rise in SHBG.In the second report (Crave et al, 1995), however,metformin therapy was combined with a hypocal-oric diet, and this treatment regimen was notassociated with any significant change in total orfree testosterone or SHBG after a 4 month period.Interpretation of this latter study is difficult, how-ever, because a parallel group of PCOS womenwho were treated with a placebo and diet didexperience a significant reduction in free testoster-one and rise in SHBG. One would have expectedthe PCOS women treated with metformin and dietto have responded at least similarly to, if not moredramatically than, the group treated with placeboand diet.

Most recently, we tested the hypothesis that tworeported features of PCOS, namely, hyperinsulin-aemic insulin resistance and increased ovariancytochrome P450cl7oc activity, are pathogen-etically linked, and that hyperinsulinaemia stimu-lates P450cl7oc activity in PCOS (Nestler andJakubowicz, 1996). To accomplish this, 24 obesewomen with PCOS were enrolled into a random-ized, single-blind, and placebo-controlled study.Oral glucose tolerance tests and 24 h GnRH agonist(leuprolide) stimulation tests were performedbefore and after oral administration of either met-formin 500 mg (n = 11) or placebo (n = 13) threetimes daily for 4-8 weeks. The women were notscreened for the presence of hyperinsulinaemia,insulin resistance or increased P450cl7a activityso that the results would be applicable to anunselected and general population of womenwith PCOS.

Metformin treatment reduced serum fasting andglucose-stimulated insulin. Amelioration of hyper-insulinaemia by metformin was also associatedwith decreased ovarian P450cl7cc enzyme activity,as demonstrated by substantial reductions inthe leuprolide-stimulated serum 17oc-hydroxy-progesterone response. Moreover, metformin treat-ment was associated with a decrease in basaland leuprolide-stimulated LH, a 44% decrease inserum-free testosterone, and a 3-fold rise in SHBG.None of these variables changed in the placebogroup.

These findings suggest that hyperinsulinaemiaincreases P450cl7oc activity in obese women with

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Figure 1. Serum-free testosterone concentrations in five obese women with polycystic ovary syndrome (PCOS) before (day 0)and after (day 10) administration of diazoxide (150 mg/day) for 10 days. Shaded bars represent mean values (adapted fromNestler et al., 1989).

PCOS either by directly stimulating ovariansteroidogenesis and/or indirectly by stimulatinggonadotrophin release. Importantly from a clinicalstandpoint, they also demonstrate that the hyper-androgenism of PCOS can be substantially amelio-rated by reducing serum insulin with metformin.

Lack of effect of insulin on ovarian androgensin normal women: evidence for a PCOS gene?

In our studies performed with diazoxide, insulinsuppression was associated with a reduction infree testosterone in women with PCOS (Nestleret al, 1989) but not in normal women (Nestleret al, 1990). At least two possible interpretationsexist for these disparate results. First of all,insulin concentrations are not elevated in normalwomen. Serum insulin in normal women maysimply not be sufficiently high to stimulateovarian androgen biosynthesis, and hence insulinmay not regulate ovarian androgens under physio-logical conditions.

However, an alternate and more attractiveexplanation is that normal women lack a geneticpredisposition to the stimulatory action of insulinon ovarian androgens. That is, it seems likely

that there exists a PCOS gene or combinationof genes, which makes the ovaries of a womanwith PCOS susceptible to insulin stimulation ofandrogen production. This hypothesis wouldexplain not only the experimental data from ourlaboratory, it is also consistent with the in-vitro studies of Barbieri et al. (1986), whichdemonstrated that insulin stimulates testosteronerelease by ovarian stroma of women with PCOSbut not by ovarian stroma of normal women,and with the results of in-vivo insulin infusionstudies which showed no effect of acute hyperin-sulinaemia on circulating testosterone in normalwomen (Nestler et al, 1987; Smith et al, 1987;Stuart et al, 1987; Dunaif and Graf, 1989). Thishypothesis is further supported by the observationthat there is familial clustering of PCOS (Givens,1988), which suggests genetic inheritance, andby the possibility that premature male patternbaldness may be a clinical marker for the PCOSgene(s) in men (Carey et al, 1994). Finally, thishypothesis would also explain why not everywoman who is obese, and is therefore bydefinition hyperinsulinaemic, develops PCOS.

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Mechanisms by which insulin could affectovarian androgen production

Direct effects of insulin on ovarian androgenproduction

On the surface it may seem paradoxical that insulinshould stimulate ovarian androgen production in awoman who is otherwise 'resistant' to insulin, butseveral theoretical mechanisms exist to explainhow a woman resistant to the effects of insulin onglucose transport could nonetheless remain fullysensitive to insulin stimulation of ovarian andro-genic pathways.

The most often cited possibilities are that insulincould either cross-react with the ovarian IGF-Ireceptor or bind to hybrid insulin receptors. Webelieve these explanations to be unlikely because:(i) the elevation in circulating insulin in PCOSwomen is usually modest and overlaps substantiallywith that observed in obese healthy women; and(ii) hybrid insulin receptors have not been identifiedon human ovaries.

It has also been suggested that insulin could actindirectly by reducing intrafollicular levels of IGF-binding protein 1 (IGFBP-1), thereby increasingintrafollicular concentrations of free IGF-I. IGF-Iis a potent stimulator of LH-induced androgensynthesis by ovarian interstitial cells (Cara andRosenfield, 1988; Adashi et al, 1992), which mayin part be due to an induction of LH receptors onthese cells by IGF-I (Cara et al, 1990). However,as reviewed elsewhere (Buyalos, 1994), thisexplanation also seems unlikely in view of evidencewhich suggests that total intrafollicular IGF-bind-ing capacity in PCOS may be increased ratherthan reduced.

We believe that insulin probably stimulatesovarian androgen production in PCOS women byactivating a signal transduction system which isdistinct and separate from the system used toenhance glucose transport. Two studies, one con-ducted in human placental cytotrophoblasts(Nestler et al, 1991b) and the other in swineovarian granulosa cells (Romero et al, 1993),support this idea by having shown that the inositol-phosphoglycan 'second messenger' system servesas the signal transduction pathway for the effectsof insulin on steroidogenesis in these tissues.The importance of these observations is that the

inositolphosphoglycan signal transduction systemmay remain intact and fully functional in conditionscharacterized by insulin resistance in terms of adefective tyrosine kinase system and impairedglucose transport. Hence, by utilizing an alternatesignal transduction pathway, the action of insulinon steroidogenesis would be preserved even in theface of glucose intolerance.

The idea that insulin stimulates ovarian androgenproduction by directly activating its own receptoris further bolstered by the report from Franks'group that steroidogenic effects of insulin aremediated by the insulin receptor itself and not bythe IGF-I receptor in primary cultures of humanovarian granulosa cells (Willis and Franks, 1995).Furthermore, using human theca cells, we recentlyprovided evidence that the inositolglycan systemserves as the signal transduction system for insulinstimulation of testosterone production by humanovarian theca cells (Nestler et al, 1997).

Indirect effects of insulin on ovarian androgenproduction

PCOS is often characterized by abnormalities inLH secretion by the pituitary. Some studies havefound that LH pulse frequency is increased inPCOS (Burger et al, 1985; Waldstreicher et al,1988; Imse et al, 1992; Berga et al, 1993), whileother studies have found no difference in LHpulse frequency between PCOS women andeumenorrhoeic women (Kazer et al, 1987; Dunaifet al, 1988; Couzinet et al, 1989). In general,however, LH pulse amplitude appears to beincreased in women with PCOS in comparisonwith healthy age- and weight-matched controlwomen (Berga et al, 1993). It is possible thatsome of the defects in LH dynamics are caused oraggravated by hyperinsulinaemia.

Insulin receptors have been identified in thehuman pituitary (Unger et al, 1991), and insulinhas been shown to modulate anterior pituitaryfunction in vitro (Yamashita and Melmed, 1986).In fact, insulin has been shown to specificallyaugment pituitary release of gonadotrophins in vitro(Adashi et al, 1981). Hence, a potential mechanismwhereby insulin could enhance ovarian androgenproduction would be by altering LH release by thepituitary. Theoretically, insulin-induced increasesin either LH pulse frequency or amplitude might

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Figure 2. Serum SHBG concentrations in six obese women with polycystic ovary syndrome (PCOS) before (day 0) and after(day 10) administration of diazoxide (150 mg/day) for 10 days. Serum androgens and oestrogens were clamped at low andconstant values throughout the study period via administration of the gonadotrophin-releasing hormone (GnRH) agonist,leuprolide. Shaded bars represent mean values. T = testosterone and E2 = oestradiol (adapted from Nestler et al, 1991).

result in enhanced ovarian androgen production.The idea that insulin enhances LH release inwomen with PCOS is supported by our recentfinding that reducing circulating insulin with met-formin leads to a decrease in basal and GnRH-stimulated LH release (Nestler and Jakubowicz,1996). Finally, our preliminary studies tend tosuggest that insulin enhances LH pulse amplitudebut does not alter LH pulse frequency in obesewomen with PCOS (J.E.Nestler, unpublished data).

Insulin and SHBGInsulin influences the clinical androgenic state notonly by directly affecting the metabolism of ovarianandrogens, but also indirectly by regulating circu-lating concentrations of SHBG. SHBG bindstestosterone with high affinity, and it is commonlyheld that it is the unbound fraction of testosterone,and not the SHBG-bound fraction, that is bioavail-able to tissues. Regulation of circulating SHBG byinsulin constitutes an important additional mechan-ism by which insulin promotes hyperandrogenism.By reducing circulating SHBG, insulin increasesthe delivery of testosterone to tissues because moretestosterone is unbound and bioavailable.

To determine whether insulin can directly influ-ence SHBG metabolism in vivo, the effect ofinsulin suppression by diazoxide on serum SHBGconcentrations was examined under conditionswhere serum androgen and oestrogen concentra-tions remained unchanged (Nestler et ah, 1991a).Ovarian steroidogenesis in six obese women withPCOS was suppressed for 2 months by the adminis-tration of a long-acting GnRH agonist. Despitesubstantial reductions in both serum androgens andoestrogens (serum testosterone concentrations fellby 82%), serum SHBG concentrations did notchange. In contrast, when diazoxide was thenadministered for 10 days to inhibit insulin release(while concurrently continuing GnRH treatment),serum SHBG concentrations rose significantly froma mean value of 17.8 ± 2.6 nmol/1 to 23.5 ± 2.0nmol/1 (P <0.003; Figure 2). Because ovariansteroidogenesis was suppressed in these women,diazoxide treatment did not alter serum androgenor oestrogen concentrations. Diazoxide does notaffect SHBG production by cultured HepG2 cells(S.R.Plymate, personal communication, 1990),nor does it alter serum SHBG values of non-

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obese healthy women with normal concentrationsof circulating insulin (Nestler et al, 1990). Thus,these observations suggest that the rise in serumSHBG concentrations following the administrationof diazoxide was due to suppression of insulinrelease, and that hyperinsulinaemia can reduceserum SHBG values in obese women withPCOS, independently of any effect on serumsex steroids.

More recently, results of in-vivo studies suggestthat insulin regulates SHBG not only in obesewomen with PCOS but in normal men and womenas well (Peiris et al, 1993; Preziosi et al, 1993;Strain et al, 1994). The results of these studiessuggest that regulation of SHBG metabolism byinsulin may be a generalized physiologicalphenomenon, and that SHBG may serve as abiological marker for hyperinsulinaemic insulinresistance in humans (Nestler, 1993).

Therapeutic implicationsThe causal role of insulin in the hyperandrogenismof PCOS has important clinical therapeutic implica-tions. It predicts that free testosterone concentra-tions in women with PCOS could be reduced bylowering circulating insulin through weight loss,accomplished either through diet or weight reduc-tion surgery. This idea is supported by two reports(Kiddy et al, 1992; Crave et al, 1995), whichshowed that serum SHBG rose and free testosteronefell in PCOS women who experienced modestweight loss by dieting. These findings constitutestrong evidence that dietary therapy is overlookedas a viable treatment option for women with PCOS.It is also possible that weight loss would not berequired to improve reproductive function, and thatdietary modification designed to decrease overallinsulinaemia might suffice. For example, byingesting more complex carbohydrates in smalleramounts rather than intermittent large binges offood.

Medications which reduce circulating insulinlevels, such as metformin (Velazquez et al, 1994;Nestler and Jakubowicz, 1996) or troglitazone(Dunaif et al, 1996), might also prove to beeffective therapies for the hyperandrogenism ofPCOS.

ConclusionsCollectively, experimental evidence suggests thathyperinsulinaemia can produce hyperandrogenismin women with PCOS via two distinct and inde-pendent mechanisms: (i) by increasing circulatingovarian androgens; and (ii) by directly reducingserum SHBG concentrations. The net result of theseactions is to increase circulating free testosteroneconcentrations. It appears likely that an inherent(probably genetically determined) ovarian defectneeds to be present in women with PCOS, whichmakes the ovary susceptible to insulin stimulationof androgen production. Limited evidence suggeststhat hyperinsulinaemia might also promote ovarianandrogen production by influencing pituitaryrelease of gonadotrophins. This latter possibility,however, has not been critically evaluated.

The clinical implication of these findings is thatamelioration of hyperandrogenism in women withPCOS may be achieved by interventions whichimprove insulin sensitivity and reduce circulatinginsulin. Such measures might include, but are notlimited to, weight loss, dietary modification, andinsulin-sensitizing medications.

AcknowledgementsStudies from the Medical College of Virginia wereperformed with the invaluable assistance of Drs JohnClore and William Blackard, past/present endocrinefellows, and General Clinical Research Center staff.They were supported by NIH Grants RO1AG11227,RO1CA64500 and RR-00065, and grants from theAmerican Diabetes Association and the Thomas F. andKate Miller Jeffress Memorial Trust.

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